Applications of gene therapy for the treatment of metabolic disorders, including cystic fibrosis, hold tremendous promise for medicine in the future. The potential effectiveness of these protocols depends, in part, on the safety, efficiency, and stability of the gene transfer vehicle. Research with recombinant adeno-associated virus over the last decade strongly suggests this novel system is well tailored for human gene therapy; wild type AAV is non pathogenic, capable of undergoing homologous recombination with host DNA, and is easily deleted of all viral genes without disrupting the latent phase of the life cycle. Despite these compelling attributes, low production yields and a limited packaging capacity stand as formidable barriers. The overall goal of this application is to develop a surrogate delivery vehicle for recombinant AAV. The prototype design takes advantage of the high titer and expanded packaging potential of recombinant adenovirus vectors; rAAV is cloned into the El region of the linear adenovirus genome where it resides as a putative mobile element. In this project Dr. Fisher and colleagues will study the optimal design of the Ad.AAV vector, considering the genotype of the adenovirus carrier and the role of rep expression for mediating rescue and targeted integration of the rAAV domain to AAVS1 sites on chromosome 19. The efficacy of the system will be determined by studying the three phases of the proposed transduction mechanism in cell line and animal models; rescue of the rAAV domain (I), formation of a circular preintegration complex (II) and recombination with cell DNA (III). Each phase is represented by a distinct molecular derivative of the nascent rAAV element that will be structurally analyzed. This project should advance the existing technology for rAAV gene transfer and provide insight as to the role of duplex intermediate structures for recombination with host cell DNA.